Field of the Invention
The present invention relates to multi-source, multi-cell clusters and methods of making, using, and cryopreserving the same.
Description of Related Art
Three-dimensional (“3-D”) cell clusters exhibit properties not seen in conventional two-dimensional cell culture. Islets of Langerhans are cell clusters within the pancreas composed of a variety of cell types including alpha-, beta-, and delta-cells, and are responsible for the maintenance of blood glucose level. Lymphocyte destruction of beta-cells (insulin-producing cells) or failure to utilize insulin are the hallmark events that result in type 1 and type 2 diabetes, respectively. Isolating islets from the pancreas of donors provides tissue that can be used for research, transplantation and drug discovery in order to develop therapies for diabetes. Once isolated from their natural location within the pancreas, islets exhibit diminished survival and function, both in vitro studies and soon after transplantation. Within the pancreas, islets are immersed with their native blood supply. After isolation, diffusion becomes the primary means of oxygen, glucose, and nutrient transport into the core of isolated native islets. Empirical modeling of diffusion barriers in native, isolated islets has demonstrated that only the outermost layers of cells are exposed to glucose and sufficient oxygen levels, resulting in core cell death. Engineering optimal islets provides a means to overcome the diffusion barriers affected by islet size limitations.
While 3-D cell clusters, such as islets, can be engineered using a variety of techniques, they still have many problems when used in different applications. For example:
Non-uniform cell number and composition in each cluster
High diffusion barrier
Not compatible with the pharmaceutical industry high-throughput instruments
Not scalable to high-throughput needs
Not able to maintain long-term experiments
Improvements in micro-mold technology (US 2010/0233239; US 2013/0029875, both incorporated by reference herein in their entireties) allow the creation of high numbers of 3-D cell clusters, such as islets reaggregated from individual islets cells in the micro-mold. However, even with these advances, limitations still exist including inconsistent response between donors to test compounds, and the fact that 3-D clusters cannot be stored and shipped without significant loss of tissue. Described herein are new methods and devices that overcome these and other problems, resulting in new applications of 3-D cell clusters.